Antibodies against pneumococcal cell wall polysaccharide (C-ps), a species specific moiety, is protective against pneumococcal infection in mice. The C-ps alone is a poor immuogen and methods for preparing covalent conjugates with T-dependent proteins were sought. Since one of the major immuno determinants of the C-ps, phosphocholine, is alkaline labile, two bifunctional chemical reagents were chosen for the conjugation process that are reactive at neutral pH. Succinimidyl N-(2-haloacetyl)-Beta-alaninate iodine salt bears an SH-reactive heloacetyl and NH2-reactive N-hydroxysuccinimide ester group which could react with the amino sugar on the C-ps. Using total iodine analysis and S-35 labeled glutathione as the halo-acetylo substrate to measure derivatization, the optimal linker/C-ps binding molar ratio was shown to be 0.5% to 1%. The second cross-linking reagent tested, succinimidyl 3-(2-pyridyldithio) propionate (SPDP), similar to SIAP, has a NH2-reactive N-hydroxy-succinimide ester group that could react with C-ps. It also has an "activated" disulfide bond, (2-pyridyldithio)-propionate which undergoes thiol-disulfide interchange with SH groups on proteins. The SPDP-C-ps reaction resulted in 4% to 6% derivatization with C-ps. We plan to use bacteria toxoids as the conjugating protein and then to study the immunogenicity of the C-ps in mice. The size of the polysaccharides in an important factor in determining the immunogenicity and the ability to form conjugates with proteins. Further, low molecular weight polysaccharides can facilitate NMR studies of polysaccharide-ligand interaction. We have applied continuous sonication to polysaccharides. Results show that sonication of high molecular weight polysaccharide at optimized poser can reduce the molecular weight of a model compound, dextran from 2,000,000 to 100,000 in 5 minutes and to 10,000 in 30 minutes independent of the type of the polysaccharides tested. This finding enables us to produce chemically unaltered lower molecular size polysaccharides at designated sized.